Transcriptional regulation involves a large number of proteins or protein complexes specifically assembled at a given promoter to activate or suppress RNA synthesis. In a specific tissue or cell type, a promoter can be turned on by a sequence of specific recognition events. Transcription factors bind cis-acting regulatory sequences; these DNA binding proteins then recruit co-activator complexes and these pre-activation complexes then recruit the core transcription machinery. Such a sequential recruitment mechanism was demonstrated on the HO gene promoter during the cell cycle in yeast (Cosma et al., 1999). Similarly, a gene can be turned off by the recruitment of transcription co-repressor complexes through sequence-specific DNA binding proteins during repression involved chromatin remodeling factors that modify histones and a long term molecular memory may be established by epigenetic modification of a specific chromatin region(s) via DNA methylation.
An advance in achieving progress in understanding the area of DNA binding proteins is the chromatin immunoprecipitation (ChIP) assay. This technology enables mapping of functional DNA elements that are engaging in interactions with specific DNA binding proteins and their associated protein complexes in vivo and has been applied to many individual case studies. In principle, this approach could lend itself to high-throughput detection methods, which would open up new opportunities for systems-level approaches to gene regulatory networks.
Researcher are seeking to identify various functional DNA elements embedded in the human genome, whether or not they are involved in gene expression, DNA replication, or establishment of chromosome territories in the cell. The method ideally suited for achieving the goal is the so-called ChIP-on-Chip technology, which is the ChIP assay coupled with high throughput detection on chips containing a microarray of human promoters.
The ChIP assay has been widely used in localizing in vivo binding sites for transcription factors. Referring to FIG. 1A, briefly, cultured cells are treated with formaldehyde to induce crosslinking between DNA and bound proteins in vivo. Treated cells are disrupted and nucleoproteins are recovered. Sonication is then used to randomly shear DNA into ˜0.5 kb pieces. Because of covalent linkage induced by crosslinking, specific proteins remain associated with fragmented DNA. Specific antibodies against target proteins are used to immunoprecipitate DNA-protein complexes. Both starting and immunoprecipitated materials are analyzed by PCR using primers specific for a given DNA region(s) under investigation. A specific in vivo interaction can be inferred if immunoprecipitation results in a significant enrichment of the DNA fragment(s) in question.
The ChIP assay has been used to detect specific targets for transcription and DNA replication factors, chromatin remodeling factors, modified histones, methylated DNA, and the like. Furthermore, the assay has also been used to detect specific association of RNA binding proteins with DNA elements bridged by transcribing RNA because transcription and splicing are known to be spatially and temporarily coupled in the cell.
The ChIP-on-Chip technology has been used to address detailed mechanistic question on selected DNA target(s). However, starting and immunoprecipitated materials have to be analyzed by PCR one at a time, which requires the selection of a target set based on available functional information. Briefly, using information from sequenced and annotated yeast genomes, individual intragenic sequences are PCR-amplified and spotted on glass to form a promoter microarray. Immunoprecipitated DNA fragments are linked by ligation with a primer-landing site on both ends, thereby permitting signal amplification by PCR (i.e., ligation-mediated PCR or LM-PCR). PCR amplified and immunoprecipitated materials are finally labeled with different fluorescence dye by random priming. Pooled PCR products are then hybridized to the promoter array to detect which promoters are specifically enriched by chromatin immunoprecipitation.
Referring to FIG. 1B, the ChIP-on-Chip technology requires 108 cells in each experiment, thus precluding analysis of development, tumorgenesis and stem cells where starting materials may be limited. In addition, microarray-based approaches will face the specificity issue. A schematic description of ChIP-on-Chip is presented in FIGS. 1A-1B and a summary comparison in Table 1, below.